Tag: microscopy

Observed doctors and medical students as they learn about the workings of the clinical microbiology lab, I’m impressed by their love of the India ink test for cryptococcus. The way this test works is: Cryptococcus is a type of infectious yeast that looks a lot like Candida if you just do a gram stain. But it has a polysaccharide capsule around each cell (unless for some odd reason it isn’t producing a capsule), wider than the cell itself. So if you put Cryptococcus in a colored liquid, most famously a solution of India ink, the polysaccharide capsule shows up as a huge empty white area around the cell. Whereas with Candida, only the cell itself is white.

We apparently don’t use this test regularly anymore, but we still show it to people in case they need to know what it is.

Something about the India ink test just makes people happy. A lot of diagnostic microbiology uses techniques that were developed several generations ago, but this one is just so simple, requiring not “acid alcohol” or various toxic red and purple substances, but merely the simplest form of ink, developed millennia ago. And to use the phrase “India ink”, instead of “colloidal carbon” or something, is such an anachronism in the 21st century. Most of us last saw that phrase when reading some classic of literature like The Secret Garden or A Bear Called Paddington. And aside from the name, there’s something magical about seeing this invisible capsule appear around what seemed to be a normal yeast cell. Like lemon-juice ink made visible.

And a bonus: High-tech 3-dimensional visualization! These are 40 focal “slices” of a single cell. From Zaragoza O, McClelland EE, Telzak A, Casadevall A (2006), Equatorial ring-like channels in the Cryptococcus neoformans polysaccharide capsule.

Today’s Pubmedwhack comes from the world of unstable metals and electron microscopy. For the definition of “Pubmedwhack”, see this earlier post.

* * *

Following the invention of immunofluorescence, scientists developed other methods for using labeled antibodies to identify certain proteins or substances under a microscope.

There’s immunohistochemistry (IHC), in which the antibodies are labeled not with a fluorescent marker, but with an enzyme which produces a visible reaction in the presence of a substrate. This is less precise, but lets you see your protein of choice under visible light while also looking at the structure of the tissue. The slides labeled this way last longer instead of being quenched by the microscope’s light source.

You can also use labeled antibodies to see things under the electron microscope (immuno-electron microscopy or immuno-EM). In 1960 Rifkin et al. (1) published an image of virus particles on a cell surface, labeled with ferritin-conjugated antibodies. As you can see, instead of the labeled antibodies changing the color of a region of the cell, each individual labeled antibody is visible as a “granule”.

from Rifkin et al (1960), Nature 187:1094

This was made possible by an innovation published one year earlier, entitled Preparation of an electron-dense antibody conjugate (2). Ferritin is a small protein which just about all organisms use as an iron carrier. When “iron-loaded”, almost a quarter of its mass is iron atoms. Therefore this is an especially electron-dense molecule, visible as a dark spot under the electron microscope, as seen above. Soon, further advances let scientists see ferritin-labeled structures inside cells.

* * *

For about a decade ferritin was the label of choice for immuno-electron microscopy. Then in 1971, a new technique came along (3, 4), in which antibodies were mixed with a solution of colloidal gold until they absorbed to the metal’s surface. Gold-labeled antibodies could be separated from free antibodies by centrifugation. Immunogold is still the dominant immuno-EM staining method 40 years later.

from Faulk et al (1971), Nature New Biology 231:101

* * *

In the 1960s, other techniques were created for immuno-EM. I have almost no EM experience and don’t know the pros and cons, but clearly there was a desire to get rid of the protein element of the electron-dense antibody label, and just attach the antibody to a metal ion. The protein was unnecessarily big, and subject to denaturation. So the 1960s also saw a lot of papers using antibodies labeled with mercury (technically the diazonium salt of tetraacetoxymercuriarsanilic acid (5) and p-(aminophenyl)-mercuric acetate (6), phrases which mean little to me).

from Zhdanov et al (1965), J Histochem Cytochem 13:684

There were also studies using antibodies labeled with heavier metals. In his long career, Ludwig Sternberger and his lab (at Johns Hopkins and elsewhere) invented several microscopy techniques, with the most important probably being the horseradish peroxidase (see original paper (7), and appreciation of it as a “citation classic”).

Unfortunately, the diazotized diazothioethers are not very stable even at Dry Ice temperatures and the solid deteriorates in a few days. Therefore, it was not surprising to observe that the osmnium tetroxide-binding iower of diazothioether antibodies was unstable, even on storage in liquid nitrogen.

How did immunofluorescence begin? Who was the first scientist to use fluorescently labeled antibodies to stain cells under a microscope?

The answer to this is pretty clear. Albert Coons of Harvard Medical School, in two papers from 1942 and 1950. In this 1962 speech (published in JAMA) Wesley Spink of the University of Minnesota describes Coons’s accomplishments.

The first time fluorescent molecules were attached to antibodies was in the early 1930s, as described in a brief letter to Nature by John Marrack (1934). But at this point the antibodies are being used in suspension, for serological experiments similar to those that detect bacteria by agglutination. In this case the bacteria can be measured colorimetrically by mixing them with fluorescently labeled antibody, washing off nonspecific antibodies, and seeing if the bacteria turn pink.

In fact, here’s the entire article.

In The Demonstration of Pneumococcal Antigen in Tissues by Means of Fluorescent Antibody (1942), Coons and associates showed that fluorescein isocyanate (FIC) could be attached to antibodies and used to stain thin tissue sections on slides, in the same way many histological stains had been used in the past. Rabbits were immunized with pneumococcus, and their serum was conjugated with FIC. This was used to visualize the bacteria in the liver of a mouse with severe pneumococcus infection. The first immunofluorescence image ever published, I believe, was this 20-minute (!) exposure.

* * *

But the fluorescent antibody technique didn’t take off until Coons and Kaplan published the sequel eight years later. I can’t find any papers from the 1940s that cite the Coons paper and actually include fluorescent images of their own.

And what a sequel! Over 2,100 citations, according to Google Scholar’s notoriously inflated algorithm. Meanwhile PubMed’s claim of 301 is a clear underestimate. It was cited a lot.

Suddenly everyone was able to use fluorescent antibodies to find out where their antigen of choice was located inside the cell, or inside the tissue, or inside the animal. Especially after 1958 when Riggs and colleagues at the University of Kansas showed how to make FITC (fluorescein isothiocyanate) conjugates, which are more stable than FIC and don’t require phosgene for their chemical synthesis. Using thiophosgene is no picnic either, but apparently it’s less deadly.

* * *

Just as early issues of the Journal of Virology are filled with papers that show “The Ultrastructure of [Name of virus]” by taking random pictures of it with an electron microscope, it seems like it was easy to get a microscopy paper published in the early 1960s. You injected rabbits with a substance, labeled their serum with fluorescein, stained some slides, and put together a manuscript called “Demonstration of [Name of substance] in [Name of tissue] by the Fluorescent Antibody Technique”.

I decided to look through some papers that cite Coons and Kaplan (1950), to find examples of early immunofluorescence. Here are a dozen images, all from at least fifty years ago.

(3) Cottontail rabbit papilloma, stained with rabbit serum specific for Shope papilloma virus and goat anti-rabbit (75x). (4) An H&E stain of the same slide, to show that the virus antigens are in the keratinized region. (Mellors, Cancer Res 1960)

—

Embryonic chicken fibroblast infected for 10 hourswith the Rostock strain of fowl plague virus (now known to be influenza A virus H7N1), stained with rabbit serum specific for “g antigen” (now called NP) (950x). The same cell stained by Giemsa-Wright stain, to show the nucleus. (Breitenfeld and Schäfer, Virology 1957)

—

Rat eye (lens and iris), stained with rabbit globulin specific for rat glomerulus (190x). Another section of the same eye stained with non-specific rabbit globulin. This is an example of the papers that used specific antiserum to find common antigens in seemingly unrelated tissues, in this case kidney and eye.(Roberts, Br J Ophthalmol 1957)

—

Human tissues, stained with human serum specific for blood group A or B. Yes, human serum. A volunteer of blood group A was used to get the serum against blood group B, and vice versa. (Szulman, J Exp Med 1960)

Kidney of rat injected with nephrotoxic rabbit antibodies to cause nephritis; the rabbit antibodies concentrate in the glomeruli, as seen by staining with goat globulin specific for rabbit globulin (37x and 205x). (Ortega and Mellors, J Exp Med 1956)

I keep having ideas for “picture gallery” posts, involving different papers’ illustrations of the same sort of thing. So far I’ve only done one (the cockroach feeding apparati) as they turn out to be a lot of work and you can never tell which papers are going to have illustrations and which ones aren’t.

In searching for pictures of encapsulated roundworm larvae in various organs of various animals, I determined that they all look sort of the same. And it’s not that interesting to compare, say, a picture of mouse muscle to a picture of raccoon or porcupine muscle. It’s all muscle. Also it may not be a good idea to extend this blog into worms and other complicated parasite species. Malaria is a parasite and also an infectious disease. But what about worms? So in the area of parasitology, I may want to stick to protozoans.

Leucocytozoons are single-celled organisms that infect birds, and are transmitted by the bugs known as blackflies. They were first observed in the 19th century in owl blood, by a zoologist named Danilewsky working in Kharkov, Ukraine. Danilewsky named them for their resemblance to white blood cells, though the exact genus “Leucocytozoon” was not applied until 1904, as detailed in this historical report by Lithuanian pedant and protozoan expert Gediminas Valkiūnas.

They seem to have life cycles similar to malaria, being fellow members of the phylum Apicomplexa. They go through many stages of life. Sporozoites are generated in the gut of an insect, and migrate to the salivary glands. The insect injects them into the blood of a vertebrate, and they go through several more stages, first in the liver and then in red blood cells. Then an insect takes a blood meal, the parasites end up in the insect’s gut, and they eventually make more sporozoites. It seems that Leucocytozoons are not as specific as malaria parasites, as they often infect white blood cells as well as RBCs, and the sporozoites of some species thrive in places other than the liver.

* * *

This figure shows the “macrogametocyte” stage (macro-gamete-ocyte) of the parasite life cycle. The macrogametocytes grow inside red blood cells, eventually filling and distorting the cells. This may be a pretty standard illustration. But it brought something else to mind.

As rendered by the Canadian authors of the paper linked above, these jaunty, big-eyed, dancing blobs are quite evocative. The genus ranges from L. vandenbrandeni (#2), which looks like a baleful ocean sunfish and lives inside the similarly aquatic birds called cormorants, to L. bonasae (#19), which infects grouse and appears to be wearing one of those baggy vinyl baseball caps from the golden age of breakdancing.

The Leucocytozoons bring to mind another type of sinister creature on a more macroscopic scale, from one of my favorite children’s books.

Yes, the Vermicious Knids, which menace visitors to the Space Hotel in Roald Dahl’s Chocolate Factory sequel, Charlie and the Great Glass Elevator. Described initially as resembling eggs with no features other than eyes, they then show that they could change shape.

“They’re tremendously proud of being able to write like that.” “But why say scram when they wanted to catch us and eat us?” “It’s the only word they know.”

The macrogametocytes of Leucytozoons look especially similar to Vermicious Knids when the latter are only displaying one eye, as in the above message to humanity, or in these creative illustrations by comics artist Isaac Cates.

Noncanonical Vermicious Knids

* * *

As you’d expect, most useful photos of these organisms are in color and therefore don’t look much like these ink drawings.

But here’s some beautiful drawings, in color, of red blood cells containing a similar parasite, Haemoproteus syrnii. Just like the first Leucocytozoons ever observed, this organism infects owls. What are they trying to say? Are they communicating by Braille or some other pattern-based system? Or by spelling out letters? They seem only capable of “C” and “O”.